Imbalances in the essential mineral elements copper, magnesium, and zinc have been demonstrated in a number of immunologically related diseases. The role of zinc in T-cell functions, specifically helper T-cell activity, has been the focus of much work. Problems in interpreting these data developed, since immune cells from deficient animals are cultured in media containing zinc when the assays for immunological reactivity are done. We developed an in vitro culture method that permits us to control the elemental environment of the immune cells. Our goal is to study specific mechanisms dependent on the three minerals in the generation of cytotoxic T lymphocytes (CTL). To accomplish this aim, we use in vitro alloantigen-stimulated mixed lymphocyte cultures as we study the involvement of copper, magnesium, and zinc in three phases of CTL generation: initiation, proliferation, and lytic phase. Studies focused on determining at what step and at what level of mineral deficiency CTL generation is modified by the lack of a metal. Results from experiments on the initiation and proliferation phases indicate that relatively high levels of the three minerals are needed and that alloantigen recognition and processing by accessory cells are a critical metal-dependent link. We have been comparing CTL generation in splenocytes from mice dietarily deficient in copper, magnesium, or zinc with CTL generation in splenocytes made deficient in vitro and have seen changes at T-lymphocyte subpopulations and function with dietary deficiencies that are not directly relatable to the mineral lack. Our studies on accessory cell function under in vitro mineral-deficient conditions demonstrated striking effects of magnesium deficiency and zinc deficiency. Morphological studies using scanning electron microscopy have also shown rapid changes in cell structure. Finally, we have been comparing production of interleukin 1 and interleukin 2 in dietary-deficient animals and in vitro-deficient conditions and have demonstrated a marked effect of magnesium and zinc on monokine/lymphokine production. (LB)